Phenyl-triazole compounds for PDE-IV inhibition

ABSTRACT

Novel compounds which are effective PDE IV inhibitors are disclosed. These compounds have the general structure of:                    
     where R 1 , R 2 , R 3 , R 4 , X 1 , X 2  and Z are described herein. The compounds possess improved PDE IV inhibition as compared to rolipram as well as with improved selectivity with regard to PDE III inhibition. Pharmaceutical compositions containing the same and methods of treatment are also disclosed.

This application is a continuation of U.S. Ser. No. 09/290,346, filedApr. 12, 1999 issued as U.S. Pat. No. 6,103,749, which is a continuationof U.S. Ser. No. 08/782,502, filed Jan. 10, 1997, issued as U.S. Pat.No. 5,922,751 which is a continuation of application of U.S. Ser. No.08/486,184, filed Jun. 7, 1995, now abandoned, which is acontinuation-in-part of U.S. Ser. No.08/265,641, filed on Jun. 24, 1994,now abandoned, the disclosures of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

Asthma is a complex disease involving the concerted actions of multipleinflammatory and immune cells, spasmogens, inflammatory mediators,cytokines and growth factors. In recent practice there have been fourmajor classes of compounds used in the treatment of asthma, namelybronchodilators (e.g., β-adrenoceptor agonists), anti-inflammatoryagents (e.g., corticosteroids), prophylactic anti-allergic agents (e.g.,cromolyn sodium) and xanthines (e.g., theophylline) which appear topossess both bronchodilating and anti-inflammatory activity.

Theophylline has been a preferred drug of first choice in the treatmentof asthma. Although it has been touted for its direct bronchodilatoryaction, theophylline's therapeutic value is now believed to also stemfrom anti-inflammatory activity. Its mechanism of action remainsunclear. However, it is believed that several of its cellular activitiesare important in its activity as an anti-asthmatic, including cyclicnucleotide phosphodiesterase inhibition, tdenosine receptor antagonism,stimulation of catecholamine release, and its ability to increase thenumber and activity of suppressor T-lymphocytes. While all of theseactually may contribute to its activity, only PDE inhibition may accountfor both the anti-inflammatory and bronchodilatory components. However,theophylline is known to have a narrow therapeutic index, and a widerange of untoward side effects which are considered problematic.

Of the activities mentioned above, theophylline's activity in inhibitingcyclic nucleotide phosphodiesterase has received considerable attentionrecently. Cyclic nucleotide phosphodiesterases (PDEs) have receivedconsiderable attention as molecular targets for anti-asthmatic agents.Cyclic 3′,5′-adenosine monophosphate (cAMP) and cyclic 3′,5′-guanosinemonophosphate (cGMP) are known second messengers that mediate thefunctional responses of cells to a multitude of hormones,neurotransmitters and autocoids. At least two therapeutically importanteffects could result from phosphodiesterase inhibition, and theconsequent rise in intracellular adenosine 3′,5′-monophosphate (cAMP) orguanosine 3′,5′-monophosphate (cGMP)in key cells in the pathophysiologyof asthma. These are smooth muscle relaxation (resulting inbronchodilation) and anti-inflammatory activity.

It has become known that there are multiple, distinct PDE isoenzymeswhich differ in their cellular distribution. A variety of inhibitorspossessing a marked degree of selectivity for one isoenzyme or the otherhave been synthesized.

The structure-activity relationships (SAR) of isozyme-selectiveinhibitors has been discussed in detail, e.g., in the article ofTheodore J. Torphy, et al., “Novel Phosphodiesterases Inhibitors For TheTherapy Of Asthma”, Drug News & Prospectives, 6(4) May 1993, pages203-214. The PDE enzymes can be grouped into five families according totheir specificity toward hydrolysis of cAMP or cGMP, their sensitivityto regulation by calcium, calmodulin or cGMP, and their selectiveinhibition by various compounds. PDE I is stimulated by Ca²⁺/calmodulin.PDE II is cGMP-stimulated, and is found in the heart and adrenals. PDEIII is cGMP-inhibited, and inhibition of this enzyme creates positiveinotropic activity. PDE IV is cAMP specific, and its inhibition causesairway relaxation, antiinflammatory and antidepressant activity. PDE Vappears to be important in regulating cGMP content in vascular smoothmuscle, and therefore PDE V inhibitors may have cardiovascular activity.

While there are compounds derived from numerous structure activityrelationship studies which provide PDE III inhibition, the number ofstructural classes of PDE IV inhibitors is relatively limited. Analoguesof rolipram, which has the following structural formula:

and of RO-20-1724, which has the following sructural formula:

have been studied.

Rolipram, which was initially studied because of its activity as anantidepressant has been shown to selectively inhibit the PDE IV enzymeand this compound has since become a standard agent in theclassification of PDE enzyme subtypes. There appears to be considerabletherapeutic potential for PDE IV inhibitors. Early work focused ondepression as a CNS therapeutic endpoint and on inflammation, and hassubsequently been extended to include related diseases such as dementiaand asthma. In-vitro, rolipram, RO20-1724 and other PDE IV inhibitorshave been shown to inhibit (1) mediator synthesis/release in mast cells,basophils, monocytes and eosinophils; (2) respiratory burst, chemotaxisand degranulation in neutrophils and eosinophils; and (3)mitogen-dependent growth and differentiation in lymphocytes (The PDE IVFamily Of Calcium-Phosphodiesterases Enzymes, John A. Lowe, III, et al.,Drugs of the Future 1992, 17(9):799-807).

PDE IV (and possibly PDE V) is present in all the major inflammatorycells in asthma including eosinophils, neutrophils, T-lymphocytes,macrophages and endothelial cells. Its inhibition causes down regulationof cellular activation and relaxes smooth muscle cells in the tracheaand bronchus. On the other hand, inhibition of PDE III, which is presentin myocardium, causes an increase in both the force and rate of cardiaccontractility. These are undesirable side effects for ananti-inflammatory agent. Theophylline, a non-selective PDE inhibitor,inhibits both PDE III and PDE IV, resulting in both desirableanti-asthmatic effects and undesirable cardiovascular stimulation. Withthis well-known distinction between PDE isozymes, the opportunity forconcomitant anti-inflammation and bronchodilation without many of theside effects associated with theophylline therapy is apparent. Theincreased incidence of morbidity and mortality due to asthma in manyWestern countries over the last decade has focused the clinical emphasison the inflammatory nature of this disease and the benefit of inhaledsteroids. Development of an agent that possesses both bronchodilatoryand antiinflammatory properties would be most advantageous.

It appears that selective PDE IV inhibitors should be more effectivewith fewer side effects than theophylline. Clinical support has beenshown for this hypothesis.

Attempts have therefore been made to find new compounds having moreselective and improved PDE IV inhibition.

OBJECTS AND SUMMARY OF THE INVENTION

It is accordingly a primary object of the present invention to providenew compounds which are effective PDE IV inhibitors.

It is another object of the present invention to provide new compoundswhich act as effective PDE IV inhibitors with lower PDE III inhibition.

It is a further object of the present invention to provide new compoundswhich have a superior PDE IV inhibitory effect as compared to rolipramor other known compounds.

It is a further object of the present invention to provide new compoundswhich have a substantially equal or superior PDE IV inhibitory effect ascompared to known chemical compounds, and which exhibit surprisinglygreater selectivity with regard to their inhibitory effects.

It is another object of the present invention to provide a method oftreating a patient requiring PDE IV inhibition.

It is another object of the present invention to provide new compoundsfor treating disease states associated with abnormally highphysiological levels of cytokine, including tumor necrosis factor.

It is another object of the present invention to provide a method ofsynthesizing the new compounds of this invention.

It is another object of the present invention to provide a method fortreating a mammal; suffering from a disease state selected from thegroup consisting of asthma, allergies, inflammations depression,dementia and diseaBe states associated with abnormally highphysiological levels of cytokine.

With the above and other objects in view, the present invention mainlycomprises a compound of the formula:

wherein:

X₁ and X₂ may be the same or different and each is O or S;

R₁ and R₂ may be the same or different and each are saturated orunsaturated straight-chain or branched alkyl groups containing from 1 to12 carbon atoms, cycloalkyl and cycloalkyl-alkyl groups containing from3 to 7 carbon atoms in the cycloalkyl moiety; and aryl and arylalkylgroups preferably containing from 6 to 10 carbon atoms, which areunsubstituted or substituted by lower alkyl groups having from 1 to 3carbon atoms, one or more halogen atoms, hydroxyl groups, cyano groups,nitro groups, carboxyl groups, alkoxy groups, alkoxycarbonyl,carboxamido or substituted or unsubstituted amino groups, orheterocyclic groups containing one or more of nitrogen, oxygen and/orsulfur in the ring, or one of R₁ and R₂ are hydrogen and the otherrepresents a hydrocarbon group as set forth above;

R₃ is hydrogen, halogen or a saturated or unsaturated straight-chain orbranched alkyl group containing from 1 to 12 carbon atoms, a cydloalkyland cycloalkyl-alkyl groups containing from 3 to 7 carbon atoms in thecycloalkyl moiety; or an aryl or aralkyl group-preferably containingfrom 6 to 10 carbon atoms, which groups are unsubstituted or substitutedby one or more halogen atoms, hydroxyl groups, cyano groups, nitrogroups, carboxyl groups, alkoxy groups, alkoxycarbonyl, carboxamido orsubstituted or unsubstituted amino groups, or heterocyclic groupscontaining one or more of nitrogen, oxygen or sulfur in the ring;

Z is a bond or a bridging (linking) group containing from 1 to 3 carbonatoms, wherein one of the carbon atoms in the bridging group may bereplaced by nitrogen, oxygen or sulfur. The carbon atoms in the bridginggroup may be saturated or unsaturated and may be unsubstituted orsubstituted with one or more substituents selected from halogen atoms(e.g., chlorine, bromine, fluorine, iodine), lower alkyl groups havingfrom 1 to 3 carbon atoms, hydroxyl groups, cyano groups, carboxylgroups, alkoxy groups, carbonyl groups, alkoxycarbonyl groups, orsubstituted or unsubstituted amino groups;

R₄ is a 5-membered heterocyclic group which contains one or more ornitrogen, oxygen and/or sulfur. atoms. The heterocyclic group may beunsubstituted or substituted with one or more halogen atoms, C₁₋₄ alkylhydroxyl groups, cyano groups, nitro groups, carboxyl groups, C₁₋₄alkoxylcarbonyl groups, alkoxy groups, alkoxycarbonyl, amido,carboxamido, or substituted or unsubstituted amino groups. Theheterocyclic group may also be substituted with alkyl, cycloalkyl andcycloalkyl-alkyl groups containing from 3 to 7 carbon atoms in thecycloalkyl moiety, aryl or arylalkyl groups preferably containing fromabout 6 to about 10 carbon atoms, or heterocyclic groups containingnitrogen, oxygen or sulfur in the ring, which groups are unsubstitutedor substituted by halogen atoms, hydroxyl groups, cyano groups, carboxylgroups, C₁₋₄ alkoxy groups, alkoxycarbonyl, carboxamido or substitutedor unsubstituted amino groups, or one or more lower alkyl groups havingfrom 1 to 3 carbon atoms; provided that R₄ is other than 4-imidazolinoneor 4-pyrolidinone.

The term “lower alkyl” is defined for purposes of the present inventionas straight or branched chain radicals.having from 1 to 3 carbon atoms.

DETAILED DESCRIPTION

The compounds of the present invention, as demonstrated in the appendedexamples, are effective in the mediation or inhibition of PDE IV inhumans and other mammals. Further, these compounds are. selective PDE IVinhibitors which possess both bronchodilatory and antiinflammatoryproperties substantially without undesirable cardiovascular stimulationcaused by PDE III inhibition. Many of these compounds have asubstantially equal or superior PDE IV inhibitory effect as compared totheophylline.

The present invention is further related to a method for the treatmentof allergic and inflammatory disease which comprises administering to amammal in need thereof an effective amount of the compounds of thepresent invention.

The present invention is also related to a method for the mediation orinhibition of the enzymatic or catalytic activity of PDE IV activity inmammals, particularly humans, which comprises. administering aneffective amount of the above-described compounds of the invention to amammal in need of PDE IV inhibition.

The compounds of the present invention may find use in the treatment ofother disease states in humans and other mammals, such as in the,treatment of disease states associated with a physiologicallydetrimental excess of tumor necrosis factor (TNF). TNF activatesmonocytes, macrophages and T-lymphocytes. This activation has beenimplicated in the progression of Human Immuno-deficiency Virus (HIV)infection and other disease states related. to the production of TNF andother cytokines modulated by TNF.

In certain preferred embodiments, the compounds of the present inventioncomprise the formula:

wherein:

R₂ is hydrogen or branched or straight chain alkyl of 1-12 carbon atoms,preferably lower alkyl, most preferably methyl or ethyl, and R₁ is alkylof 1-12 carbon atoms, which may be substituted by one or more halogens,or cycloalkyl of 3-6 carbon atoms, preferably cyclopentyl which may besubstituted by R₅ as shown in the following structural formula:

wherein R₅ is hydrogen or a saturated or unsaturated straight-chainlower alkyl group containing from about 1 to about 6 carbon atoms,unsubstituted or substituted with one or more halogen atoms, hydroxylgroups, cyano groups, nitro groups, carboxyl groups, alkoxy groups,alkoxycarbonyl, carboxamido or substituted or unsubstituted aminogroups;

R₃ is hydrogen, lower alkyl or halogen;

Z is a linkage selected from a bond, —NH—, —CH₂—, —CH₂CH₂—, —CH₂NH—,CH₂N(Me), NHCH₂—, —NH—, —CH₂CONH—, —NHCH₂CO—, —CH₂CO—, —COCH₂—,—CH₂COCH₂—, —CH(CH₃)—, —CH═, and —HC═CH—;

X₁ and X₂ may be the same or different and each is O or S; and

R₄ is an unsubstituted or substituted pyrazole, imidazole, or triazoleas set forth previously.

In one preferred embodiment, R₄ is a substituted pyrazole having thefollowing structure:

wherein R₆ is a substituted or unsubstituted lower alkyl having fromabout 1 to about 3 carbon atoms.

In a particularly preferred embodiment of the present invention, thecompounds have the formula:

where X₁ and X₂ may be the same or different and each is O or S;

R₁ is alkyl of 1-12 carbon atoms or cycloalkyl of 3-6 carbon atoms,which cycloalkyl may be substituted by one or more alkyl groups or byone or more halogens;

R₂ is hydrogen or alkyl of 1-12 carbon atoms;

R₃ is hydrogen, lower alkyl or halogen;

Z is a linkage selected from a bond, —NH—, —CH₂—, —CH₂CH₂—, —CH₂NH—,NHCH₂—, —NH—, —CH₂N(Me)—,—CH₂CONH—, —NHCH₂CO—, —CH₂CO—, —COCH₂—,—CH₂COCH₂—, —CH(CH₃)—, —CH═, and —HC═CH—; and

R₇ is C or CH₂C.

One preferred compound of the present invention is3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-hydroxymethyl pyrazole.

In certain preferred embodiments, compounds of the present invention areprepared by the two-step process shown below for the preferred compoundof the invention,3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-hydroxymethyl pyrazole.

The condensation of 3-cyclopentyloxy-4-methoxybenzaldehyde with3-amino-4-pyrazolecarboxylic acid in the presence of sodiumcyanoborohydride produces3-(3-cyclopentyloxy-4-methoxybenzyl-amino)-4-pyrazolecarboxylic acid in48% yield. Treatment with excess borane-tetrahydrofuran complex intetrahydrofuran solution gives3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-hydroxymethylpyrazole(Example 1) in 40% yield.

In other embodiments where R₄ is an imidazole, triazole or pyrazole, thecompounds can be synthesized by reductively condensing3-cyclopentyloxy-4-methoxybenzaldehyde with amino heterocycles usingsodium cyanoborohydride. The reaction is shown below.

The compounds of the present invention have been found to be highlyeffective PDE IV inhibitors, the inhibition of which is in factsignificantly and surprisingly greater than that of theophylline

Thus, the concentration which yields 50% inhibition of PDE IV (IC₅₀) for3-(3-cyclopentyloxy-4-ethoxybenzylamino)-4-hydroxymethyl pyrazole is 16μm (0.016 of whereas the IC₅₀ for rolipram when run in the same assaywas 4.5 μM. Historically, the IC₅₀ for rolipram is considered to be 3.5μM. In any case, it is apparent that Example 1 of the present inventionis several hundred times as effective as a PDE IV inhibitor as comparedto rolipram.

By comparison, PDE III inhibition of Example 1 of the present inventionis within 1 order of magnitude of that of rolipram, and thereforerelative to the percentage increase in PDE IV inhibition, it is clearthat the compound of the invention is much more highly selective as aPDE IV inhibitor than rolipram.

Accordingly, the compounds of the present invention can be administeredto anyone requiring PDE IV inhibition. Administration may be orally,topically, by suppository, inhalation or insufflation, or parenterally.

The present invention also encompasses all pharmaceutically acceptablesalts of the foregoing compounds. One skilled in the art will recognizethat acid addition salts of the presently claimed compounds may beprepared by reaction of the compounds with the appropriate acid via avariety of known methods. Alternatively, alkali and alkaline earth metalsalts are prepared by reaction of the compounds of the inventionwith-the appropriate base via a variety of known methods. For example,the sodium salt of the compounds of the invention can be prepared viareacting the compound with sodium hydride.

Various oral dosage forms can be used, including such solid forms astablets, gelcaps, capsules, caplets, granules, lozenges and bulk powdersand liquid forms such as emulsions, solution and suspensions. Thecompounds of the present invention can be administered alone or can becombined with various pharmaceutically acceptable carriers andexcipients known to those skilled in the art, including but not limitedto diluents, suspending agents, solubilizers, binders, disintegrants,preservatives, coloring agents, lubricants and the like.

When the compounds of the present invention are incorporated into oraltablets, such tablets can be compressed, tablet triturates,enteric-coated, sugar-coated, film-coated, multiply compressed ormultiply layered. Liquid oral dosage forms include aqueous andnonaqueous solutions, emulsions, suspensions, and solutions and/orsuspensions reconstituted from non-effervescent granules, containingsuitable solvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, coloring agents, and flavorings agents. When thecompounds of the present invention are to be injected parenterally, theymay be, e.g., in the form of an isotonic sterile solution.Alternatively, when the compounds of the present invention are to beinhaled, they may be formulated into a dry aerosol or may be formulatedinto an aqueous or partially aqueous solution.

In addition, when the compounds of the present invention areincorporated into oral dosage forms, it is contemplated that such dosageforms may provide an immediate release of the compound in thegastrointestinal tract, or alternatively may provide a controlled and/orsustained release through the gastrointestinal tract. A wide variety ofcontrolled and/or sustained release formulations are well known to thoseskilled in the art, and are contemplated for use in connection with theformulations of the present invention. The controlled and/or sustainedrelease may be provided by, e.g., a coating on the oral dosage form orby incorporating the compound(s) of the invention into a controlledand/or sustained release matrix.

Specific examples of pharmaceutically acceptable carriers and excipientsthat may be used for formulate oral dosage forms, are described in theHandbook of Pharmaceutical Excipients, American PharmaceuticalAssociation (1986), incorporated by reference herein. Techniques andcompositions for making solid oral dosage forms are described inPharmaceutical Dosage Forms: Tablets (Lieberman, Lachman and Schwartz,editors) 2nd edition, published by Marcel Dekker, Inc., incorporated byreference herein. Techniques and compositions for making tablets(compressed and molded), capsules (hard and soft gelatin) and pills arealso described in Remington's Pharmaceutical Sciences (Arthur Osol,editor), 1553-1593 (1980), incorporated herein by reference. Techniquesand composition for making liquid oral dosage forms are described inPharmaceutical Dosaae Forms: Disperse Systems, (Lieberman, Rieger andBanker, editors) published by Marcel Dekker, Inc., incorporated hereinby reference.

When the compounds of the present invention are incorporated forparenteral administration by injection (e.g., continuous infusion orbolus injection), the formulation for parenteral administration may bein the form of suspensions, solutions, emulsions in oily or aqueousvehicles, and such formulations may further comprise phartaceuticallynecessary additives such as stabilizing agents, suspending agents,dispersing agents, and the like. The compounds of the invention may alsobe in the form of a powder for reconstitution as an injectableformulation.

The dose of the compounds of the present invention is dependent upon theaffliction to be treated, the severity of the symptoms, the route ofadministration, the frequency of the dosage interval, the presence ofany deleterious side-effects, and the particular compound utilized,among other things.

The PDE IV inhibitory compounds of the present invention may be examinedfor their PDE IV inhibitory effects via the techniques set forth in thefollowing examples, wherein the ability of the compounds to inhibit PDEIV isolated from bovine tracheal smooth muscle is set forth.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples illustrate various aspects of the presentinvention, and are not to be construed to limit the claims in any mannerwhatsoever.

EXAMPLE 1 3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-hydroxymethylpyrazole

The above-mentioned compound was prepared by a two-step process as setforth below:

A: 3-(3-cyclopentyloxy-4-methoxy-benzylamino)-4-pyrazole-carboxylic acid

A suspension of 3-amino-4-pyrazole carboxylic acid (3.56 g, 28 mmol) in400 ml of methanol and 3-cyclopentyloxy-4-methoxybenzaldehyde (5.06 g,23 mmol) was stirred at room temperature for 16 hours with sodiumcyanoborohydride, 1.85 g (95% pure, 28 mmol). The methanol wasevaporated under reduced pressure and the residue was taken up in 100 mlof 15% sodium hydroxide and-extracted twice with 100 ml portions ofether. The aqueous layer was acidified to pH 5 with 5N hydrochloric acidand extracted twice with 100 ml portions of ethyl acetate. The ethylacetate was dried over sodium sulfate and evaporated. The residue wastriturated with ether to give 3.7 g of3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-pyrazolecarboxylic acid(48.7%). Pure 3-(3-cyclopentyloxy-4-methoxybenzylamino)pyrazolecarboxylic acid shows mp. 134-136° C. after crystallization frommethanol.

B: 3-(3′-cyclopentyloxy-4-methoxybenzylamino)-4-hydroxymethyl-pyrazole

Pure 3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-pyrazolecarboxylic acid(compound 3) (1.32 g, 4.0 mmol) was dissolved in 50 ml of THF and cooledto −15° C. under nitrogen. Borane-tetrahydrofuran (30 ml of 1M solution,30 mmol) was added over 30 minutes and the resulting solution wasstirred for 72 hours at room temperature. Methanol (30 ml) was added tothe reaction and the solvent was evaporated under reduced pressure. Theresidue was treated with 160 ml of methanol and evaporated again. Thecrude product was treated with 30 ml of 10% aqueous ammonia and somebrine and then extracted with three 50 ml portions of ethyl acetate.Evaporation of the solvent afforded 1.1 g of crude3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-hydroxymethylpyrazole whichwas purified by flash chromatography on 30 g of flash chromatographysilica gel. Elution with ethyl acetate/hexane (1:4) afforded a smallamount of material which was discarded. Elution with 60 ml of 1:1 ethylacetate/hexane gave 600 mg of3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-hydroxymethylpyrazole whichwas recrystallized from 2 ml of toluene to give 500 mg of pure3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-hydroxymethylpyrazole(compound 4) mp 129-130° C.

EXAMPLE 23-(3-Cyclopentyloxy-4-methoxybenzyl-amino)-4-pyrazolecarboxamide

To a stirred suspension of 3-amino-4-pyrazolecarboxamide hemisulfate(0.005 mole) was added 0.14 g of potassium hydroxide (KOH),3-cyclopentyloxy-4-methoxybenzaldehyde (0.88 g, 0.004 mole), and 95%sodium cyanoborohydride (0.33 g, 0.004 mol). Stirring was continued atroom temperature for 24 hours after which KOH (0.56 g) was added and thesolvent was evaporated under reduced pressure. The residue was treatedwith 50 ml of brine and extracted twice with 50 ml portions of ethylacetate. The ethyl acetate was evaporated under reduced pressure and theresidue was triturated with ether to give 0.4g of the crude titlecompound. Crystallization from 15 ml of acetone gave 0.25 g (15%). ofthe pure title compound, mp 128-130° C.

EXAMPLE 3 3-(3-Cyclopentyloxy-4-methoxybenzylamino)-1,2,4-triazole

To a stirred solution of 3-amino-1,2,4-triazole (1.5 g, 0.018 mol), 7.5ml of 1N HCl, and 50 ml of methanol was added3-cyclopentyloxy-4-methoxybenzaldehyde (3.3 g, 0.015 mol) and sodiumcyanoborohydride (0.945 g, 0.015 mol). A precipitate formed slowly andafter 20 hours the precipitate was filtered and recrystallized frommethanol (135 ml) to give 1.0 g (0.0034 mol, 23%) of the title compound,mp 203-204° C.

EXAMPLE 4 3-(3-Cyclopentyl-oxy-4-methoxybenzylamino)pyrazole

To a solution of 3-aminopyrazole (1.5 g, 0.018 mol) in 50 ml of methanoland 7.5 ml of 1N aqueous HC1 was added3-cyclopentyloxy-4-methoxybenzaldehyde (3.3 g, 0.015 mol) and 95% sodiumcyanoborohydride (1.0 g, 0.015 mol). The solution was stirred at roomtemperature for 20 hours after which concentrated aqueous hydrochloricacid (Hcl) was added to pH 2. The solvent was evaporated at reducedpressure and the residue was extracted twice with 50 ml portions ofether. The ether extracts were discarded and the aqueous solution wasbrought to pH 10 with 5N aqueous NaOH. The mixture was extracted twicewith 50 ml portions of ethyl acetate. The combined ethyl acetateextracts were dried (MgSO₄) and evaporated. The solid residue wastriturated with ether to give the pure title compound (1.3 g, 30%), mp106-110° C.

EXAMPLE 5 5-(3-Cyclopentyloxy-4-methoxybenzylamino)-1,2,3-triazole

To a stirred solution of 5-amino-1,2,3-triazole (3.0 g, 0.036 mol) in100 ml of methanol and 15 ml of 1N HCl was added sodium cyanoborohydride(1.89 g, 0.030 mol) and 3-cyclopentyloxy-4-methoxybenzaldehyde (6.6 g,0.30 mol). The reaction mixture was stirred at room temperature for 64hours and then acidified to pH 2 with concentrated HCl. The solution wasevaporated at reduced pressure and the residue was treated with water(100 ml) and extracted twice with 100 ml portions of ethyl acetate. Theethyl acetate extracts were combined and extracted twice with 50 mlportions of 3N HCl and then discarded. The aqueous extracts werecombined with the original aqueous layer and basified to pH 12 withpotassium hydroxide pellets. The alkaline solution was extracted twicewith 100 ml portions of ethyl acetate. The ethyl acetate extracts werecombined and evaporated to give a solid which was crystallized fromethyl acetate to give 2.9 g (34%) of the title compound mp 113-114° C.collected in two crops.

EXAMPLE 6 2-(3-Cyclopentyloxy-4-methoxybenzylamino)imidazole

A stirred suspension of 2-aminoimidazole sulfate (5.67 g, 0.043 mol) in100 ml of methanol was treated with solid KOH (0.65 g, 0.011 mol)followed by 3-cyclopentyloxy-4-methoxybenzaldehyde (7.26 g, 0.033 mol).To this mixture was added dropwise over 30 minutes solution of 95%sodium cyanoborohydride (2.2 g, 0.33 mol) in methanol (30 ml). Thereaction mixture was stirred at room temperature for 64 hours afterwhich solid KOH (2.45 g, 0.055 mol) was added. After the KOH haddissolved, the mixture was filtered and the filtrate was evaporatedunder reduced pressure. The residue was treated with saturated NaCl andextracted three times with 50 ml portions of ethyl acetate. The combinedethyl acetate extracts were extracted twice with 5N aqueous HCl.Evaporation of the ethyl acetate yielded 5.5 g of unreacted3-cyclopentyloxy-4-methoxybenzaldehyde. The aqueous HCl extract wasbasified to pH 12 with solid KOH and extracted twice with 50 ml portionsof ethyl acetate. Evaporation of the ethyl acetate afforded 2.4 g of abrown oil which was purified by flash chromatography over 40 g of flashchromatography silica gel. Elution with methylene chloride-methanolmixtures afforded 1.8 g of the title compound eluted with 5%methanol-methylene chloride. The 1.8 g of material was treated withsolution of picric acid (5 g, 0.021 mol) in 50 ml of ethanol. Thepicrate (1.9 g) was collected and recrystallized from 25 ml of ethanolto give 1.7 g of the pure picrate. The picrate (1.5 g) was dissolved in200 ml of ethyl acetate and extracted 5 times with 25 ml portions of 2Naqueous LiOH. The ethyl acetate solution was evaporated and the residue(0.9 g) was purified by flash chromatography over 40 g of silica gel,elutinq with methanol-methylene chloride mixtures to give 0.5 g of thematerial which was crystallized from 15 ml of ethyl acetate to give 0.3g (3%) of the pure title compound, mp 114-115° C.

EXAMPLE 73-(3-Cyclopentyloxy-4-methoxybenzyl-amino)-4-pyrazolecarboxylic acid

To a suspension of 3-aminopyrazole-4-carboxylic acid (3.56 g, 0.023 mol)in 400 ml of methanol was added 3-cyclopentyloxy-4-methoxybenzaldehyde(5.06-g, 0.023 mole) and 95% sodium cyanoborohydride (1.85 g, 0.028mol). The reaction mixture was stirred for 16 hours and then themethanol was evaporated under reduced pressure. The residue was treatedwith 100 ml of 15% aqueous NaOH and extracted twice with 100 ml portionsof ether. The aqueous layer was acidified to pH 5 with 5N aqueous HCland extracted twice with 100 ml portions of ethyl acetate. The ethylacetate extracts were evaporated under reduced pressure and the residuewas triturated with ether to give 3.7 g (49%) of the title compound, mp134-136°, after recrystallization from methanol.

EXAMPLE 8 5-(3-Cyclopentyloxy-4-methoxybenzal)hydantoin

To a stirred solution of diethyl 5-hydantoylphosphonate (10 g, 42.3mmol) and triethylamine (5.70 g, 56 mmol), in dry acetonitrile (100 ml),at room temperature was added a solution of3-cyclopentyloxy-4-methoxybenzaldehyde (8 g, 36.3 mmol) in dryacetonitrile (75 ml), dropwise over 30 minutes. The resulting mixturewas stirred at room temperature overnight, after which volatiles wereremoved in vacuo. The residue was diluted with 0.05N HCl (120 ml) andthe precipitate collected by filtration. Recrystallization of thismaterial with methylene chloride/methanol/diethyl ether afforded thetitle compound as a pale yellow solid (8.54 g), m.p. 220-222° C.

¹H NMR (CDCl₃; 250 MHz) δ 7.72 (brs; 1H), 7.66 (brs, 1H), 6.90 (m, 3H),6.66 (s, 1H), 4.79 (m, 1H), 3.88 (s, 3H), 1.84 (m, 6H), 1.62 (m, 2H).

EXAMPLE 9 4-(3-Cyclopentyloxy-4-methoxybenzyl)-5-methyl-pyrazol-3-one

A. (E)/(Z)Ethyl2-acetyl-3-(3-cyclopentyl-oxy-4-methoxylphenyl)-2-propenoate

To a stirred solution of 3-cyclopentyloxy-4-methoxybenzaldehyde (4.0 g,22.69 mmol) and ethyl acetoacetate (2.95 g, 22.67 mmol) in benzene (60ml) were added piperidine (104 mg, 1.22 mmol) and glacial acetic acid(286 mg, 4.76 mmol) in one portion. The resulting solution was refluxedat 85° C. for 3 hours with continuous removal of water. Furtherpiperidine (208 mg, 2.44 mmol) and glacial acetic acid (858 mg, 14.28mmol) were added and refluxing continued for a further 5 hours. Themixture was cooled to room temperature and diluted with diethyl ether(300 ml). The organics were washed successively with 5% aqueous HCl (100ml) and water (2×100 ml). The resulting etheral extracts were dried(Na₂SO₄) and concentrated in vacuo to give a deep orange oil. The oilwas purified by flash chromatography (SiO₂; ethyl acetate/hexane (3:7))to afford the title compound as an orange oil (983 mg).

¹H (CDCl₃; 250 MHz) 6 7.57 and 7.46 (2s, 1H), 6.92 (m, 3H), 6.92 (m,3H), 4.70 (m, 1H), 4.34 and 4.26 (2q, 2H), 3.87 and 3.86 (2s, 3H), 2.39and 2.37 (2s, 3H), 1.87 (m, 6H), 1.62 (m, 2H), 1.31 (t, 3H).

B. Ethyl 2 acetyl-3-(3-cyclorentyl-oxy-4-methoxphenyl)-propanoate

A solution of (E)/(Z) ethyl2-acetyl-3-(3-cyclopentyloxy-4-methoxyphenyl)-2-propenoate (745 mg, 2.24mmol) in ethyl acetate (20 ml) containing palladium on activated carbon(10% palladium, 25 mg) was hydrogenated at 40 p.s.i. for 5 hours at roomtemperature. The mixture was filtered through celite and the filter cakewashed with ethyl acetate (2×25 ml). The filtrate was concentrated invacuo and purified by flash chromatography (SiO₂; ethyl acetate/hexane(3:7)) to afford the title compounds as a pale yellow oil (585 mg).

R_(f) (SiO₂; ethyl acetate/hexane (3:7)) 0.45.

C. 4-(3-Cyclopentyloxy-4-methoxy-benzyl)-5-methyl-pyrazol-3-one

To a stirred solution of ethyl2-acetyl-3-(3-cyclopentyloxy-4-methoxyphenyl)-propanoate (585 mg, 1.74mmol) in absolute ethanol (10 ml) at 0° C. was added hydrazinemonohydrate (87 mg, 1.74 mmol) in one portion. The resulting solutionwas stirred at 0° C. for 45 minutes and then refluxed at 80° C. for 3hours. After cooling to room temperature, the reaction mixture wasconcentrated in vacuo and purified by flash chromatography(SiO₂;methylene chloride/ethanol/ammonia (4:1:0.1)) to-afford the titlecompound as a white solid (343 mg). m.p. 186-187° C.

¹H NMR (CD₃OD; 250 MHz) δ 6.78 (m, 3H), 4.73 (m, 1H), 3.76 (s, 3H), 3.56(s, 2H), 2,03 (s, 3H), 1.80 (m, 6H), 1.59 (m, 2H).

EXAMPLE 104-[1-(3-Cyclopentyloxy-4-methoxy-phenyl)ethyl]-5-methylpyrazol-3-one

A.N-Methoxy-N-methyl-3-(3-cyclopentyloxy-4-methoxyphenyl)-prot-2-enamide

To a stirred solution ofdiethyl(N-methoxy-N-methyl-carbamoylmethyl)phosphonate (2.60 g, 10.90mmol) in dry tetrahydrofuran (50 ml) at −78° C. was added lithiumdiisopropylamide (1.5M solution in cyclohexane, 7.33 ml, 11 mmol)dropwise over 3 minutes. The resulting solution was stirred at −78° C.for 30 minutes, after which time 3-cyclopentyloxy-4-methoxybenzaldehyde(1.5 g, 6.81 mmol) in dry tetrahydrofuran (5 ml) was added in oneportion. The reaction was allowed to warm to room temperature andstirring continued overnight. After which time the reaction mixture wasdiluted with water (25 ml) and the tetrahydrofuran removed in vacuo. Theaqueous residue was diluted with brine (25 ml) and extracted withmethylene chloride (3×60 ml). The organic extracts were dried (Na₂SO₄)and evaporated in vacuo to give a yellow liquid. The liquid was purifiedby flash chromatography (SiO₂; hexane/ethyl acetate (65:35)) to affordthe title compound as a pale yellow liquid (1.986 g).

R_(f) (SiO₂; ethyl acetate/hexane (35:65)) 0.15

B. (E)-4-(3-Cyclopentyloxy-4-methoxyrhenyl)-3-buten-2-one

To a stirred solution ofN-methoxy-N-methyl-3-(3-cyclopentyloxy-4-methoxyphenyl)prop-2-enamide(1.986 g, 6.50 mmol) in dry tetrahydrofuran (15 ml) at −78° C. was addedmethyl lithium (1.4M solution in hexanes, 10.20 ml, 14.3 mmol) dropwiseover 3 minutes. The resulting solution was stirred for 1 hour at 0° C.,after which time the reaction mixture was diluted with 5% HCl inmethanol (20 ml) at 0° C., and partitioned between brine (35 ml) and anequal volume of methylene chloride and diethyl ether (100 ml). Theorganics were dried (Na₂SO₄) and concentrated in vacuo to give a lightyellow oil. The oil was purified by flash chromatography (SiO₂; ethylacetate/hexane (35:65)) to afford the title compound as a colorless oil(1.642 g).

¹H NMR (CDCl₃; 250 MHz) δ 7.43 (d, J=16.2 Hz, 1H), 7.08 (m, 2H), 6.85(d, 1H), 6.57 (d, J=16.2 Hz, 1H), 4.78 (m, 1H), 3.87 (s, 3H), 2.36 (s,3H), 1.89 (m, 6H), 1.62 (m, 2H).

C.[[4-(3-Cyclopentyloxy-4-methoxyphenyl)-2-penten-2-yl]oxy]tributylsilane

To a stirred suspension of cuprous iodide (1.15 g, 6.045 mmol) in drytetrahydrofuran (10 ml) at 0° C. was added methyl lithium (1.4M solutionin hexanes, 8.635 ml, 12.09 mmol) dropwise over 3 minutes. The resultingsolution was stirred at 0° C. for 30 minutes, then cooled to −78° C. andchlorotributylsilane (3.54 g, 15.07 mol) in dry tetrahydrofuran (5 ml)added so that the temperature of the reaction mixture did not rise above−60° C. After addition was complete the reaction was stirred at −78° C.for 10 minutes then(E)-4-(3-cyclopentyloxy-4-methoxyphenyl)-3-buten-2-one (787 mg, 3.02mmol) in dry tetrahydrofuran (5 ml) added dropwise over 3 minutes. Theresulting solution was stirred at −78° C. for 45 minutes and quenched at−78° C. by the addition of aqueous saturated NaHCO₃ (25 ml) and water(20 ml).

After warming to room temperature, the precipitate was removed byfiltration and the filtrate extracted with pentane (3×70 ml). Theorganics were washed with water (20 ml), brine (20 ml) and dried(Na₂SO₄) and concentrated in vacuo to give an oil. The oil was purifiedby flash chromatography (SiO₂;1) hexane 2) hexane/ethyl acetate (9:1))to, afford the title compound (contaminated withtributylsilane/chlorotributylsilane) as a colorless oil (3.33g). R_(f)(SiO₂; hexane/ethyl acetate (9:1)) 0.42

D. Methyl 2-acetyl-3-(3-cyclopentyloxy-4-methoxyphenyl)-butanoate

To a stirred solution of[[4-(3-(cyclopentyloxy-4-methoxyphenyl)-2-penten-2-yl]oxy]tributylsilaneprepared as detailed above, (3.38 g, containing approximately 3.0 mmolof the desired compound and 8.0 mmol ofchlorotributylsilane/tributylsilane) in dry tetrahydrofuran (10 ml) at0° C. was added methyl lithium (1.4M in diethyl ether, 9.37 ml, 13.12mmol) dropwise over 3 minutes. The resulting clear solution was stirredfor 18 hours at room temperature, then cooled to −78° C.Hexamethylphosphoramide (537 mg, 3.0 mmol) and methylcyanoformate (280mg, 3.3 mmol) in dry tetrahydrofuran (3 ml) were added and the mixturestirred at −78° C. for 30 minutes. After warming to 0° C. the reactionmixture was portioned between water (40 ml) and diethyl ether (100 ml).The organics were washed with bring (30 ml), dried (Na₂SO₄) andevaporated in vacuo to give an oil. The oil was purified by flashchromatography (SiO₂:hexane/ethyl acetate (85:15)) to afford the titlecompound as a colorless oil (362 mg).

R_(f) (SiO₂; ethyl acetate hexane (1:4) 0.26 and 0.22 mixture ofisomers.

E. 4-[1-(3-Cyclopentyloxy-4-methoxyphenyl)ethyl]-5-methyl-pyrazol-3-one

To a stirred solution of methyl2-acetyl-3-(3-cyclopentyloxy-4-methoxyphenyl)-butanoate (362 mg, 1.08mmol) in absolute ethanol (10 ml) at 0° C. was added hydrazinemonohydrate (54 mg, 1.08 mmol) in one portion. The resulting solutionwas stirred at 0° C. for 45 minutes and then refluxed at 80° C. for 3hours. Further hydrazine monohydrate (15.6 mg, 3.1×10⁻⁴ mol) was addedand refluxing continued for a further 3 hours. After cooling to roomtemperature, the reaction mixture was concentrated in vacuo and purifiedby flash chromatography (SiO₂; methylene chloride/ethanol/ammonia(97.5:2.5:0.25)) to afford the title compound as a white solid (76 mg).

¹H (CDCl₃; 250 MHz) δ 6.80 (m, 3H), 4.72 (m, 1H), 3.93 (q, 1H), 3.79 (s,3H), 2.00 (s, 3H), 183 (m, 6H), 1.60 (d, 3H), 1.57 (m, 2H).

EXAMPLE 11 5-(3-Cyclopentyloxy-4-methoxybenzal)hydantoin

A solution of 3-(3-cyclopentyloxy-4-methoxybenzal) hydantoin (1 g, 3.30mmol) in ethanol/methylene chloride ((3:1) 50 ml) containing palladiumon activated carbon (5% Pd, 200 mg) was hydrogenated at 40 p.s.i. for 6hours at room temperature. The mixture was filtered through celite andthe filter cake washed with methylene chloride (2×40 ml). The filtratewas concentrated in vacuo and purified by flash chromatography (SiO₂;methylene chloride/ethanol/ammonia (96:4:0.4)) to afford the titlecompound as a white solid (578 mg). m.p. 151-153° C.

¹H NMR (CDCl₃; 250 MHz) 6 8.41 (brs, 1H), 6.74 (m, 3H), 5.79 (brs, 1H),4.73 (brs, 1H), 4.24 (dd, 1H), 3.80 (s, 3H), 2.98 (AB system 2×dd, 2H),1.83 (m, 6H), 1.59 (m, 2H).

EXAMPLE 12 Ethyl3-(3-cyclopentyloxy-4-methyoxybenzylamino)pyrazole-4-carboxylate

A solution of ethyl 3-aminopyrazole 4-carboxylate (70 g, 0.45 mol) and3-cyclopentyloxy-4-methoxybenzaldehyde (100 g, 0.45 mol) in methanol(400 mL) was treated with sodium cyanoborohydride (19.0 g, 0.30 mol) andsodium acetate (2.0 g, 0.024 mol) in a stirred reaction vessel equippedwith pH stat attached to a syringe pump and a syringe filled with 3 Nacetic acid in methanol. The pH stat was set to maintain pH 7. Theacetic acid solution was added as the reaction proceeded. After 24hours, 169 mL of acetic acid solution had been added and no aldehyderemained- in the reaction mixture by TLC (silica gel, 10%methanol/methylene chloride) and 3-cyclopentyloxy-4-methoxy-benzaldehyde(19.0 g, 0.086 mol) was added to the reaction mixture. After 30 hours, atotal of 216 mL of acetic acid solution had been added and3-cyclopentyloxy-4-methoxybenzaldehyde (16 g, 0.072 mol) was added tothe reaction mixture. After 48 hours, 263 mL of acetic acid solution hadbeen added and additional 3-cyclopentyloxy-3-methoxybenzaldehyde (29 g,0.13 mol) was added to the reaction mixture. After 76 hours, 320 mL ofacetic acid solution had been consumed and the reaction mixture wasevaporated under reduced pressure. The residue was partitioned betweenwater (400 mL) and ethyl acetate (400 mL) and the ethyl acetate wasevaporated under reduced pressure to give 240 g of crude product. Thecrude product was purified by flash chromatography over 900 g of flashchromatography silica gel and eluted with methanol-methylene chloridemixtures. Elution with pure methylene chloride afforded by-product3-cyclopentyloxy-4-methoxybenzyl alcohol. Elution with 1-25%methanol-methylene chloride gave 91 g (42%) of nearly pure ethyl3-(3-cyclopentyloxy-4-methoxybenzyl-amino)pyrazole-4-carboxylate. Aportion of this ethyl 3-(3-cyclopentyloxy-4-methoxybenzylamino)pyrazolecarboxylate (29 g) was recrystallized from methyl t-butyl ether to give16.8 g of pure ethyl 3-(3-cyclopentyloxy-4-methoxybenzyl-amino)pyrazole-4-carboxylate, mp 102-104°.

EXAMPLE 13 3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-cyanopyrazole

A solution of 3-amino-4-cyanopyrazole (5.0 g, 0.16 mol),3-cyclopentyloxy-4-methoxybenzaldehyde (10.0 g, 0.045 mol) and sodiumacetate (0.2 g, 2.4 mol) in methanol (70 mL) was placed in a stirredreaction flask equipped with a pH stat controlling a syringe pump andsyringe filled with 3 N acetic acid in methanol. The pH stat was set tomaintain pH 7. After 24 hours, more3-cyclopentyloxy-4-methoxybenzaidehyde (7.0 g, 0.031 mol) was added andthe reaction was continued. After 48 hours, more3-cyclopentyloxy-4-methoxybenzaldehyde (4.0 g, 9.0 mmol) and sodiumcyanoborohydride (0.5 g, 7.9 mmol) was added and the reaction wascontinued. After 72 hours, more sodium cyanoborohydride (0.5 g, 7.9mmol) was added and after 96 hours, the reaction was stopped log and thesolvent was removed under reduced pressure. The residue was partitionedbetween ethyl acetate (75mL) and water (50 mL). The ethyl acetate layerwas washed with water (2×50 mL) and evaporated. The crude product waschromaotgraphed over 200 g of flash chromatography silica gel. Elutionwith 2% methanol/methylene chloride removed some impurities including3-cyclopentyloxy-4-methoxybenzyl alcohol. Continued elution withmethanol/methylene chloride mixtures of increasing methanolconcentration afforded 8 g (56%) of nearly pure title compound. Pure3-(3-cyclopentyloxy-4-methoxy-benzylamino)-4-cyanopyrazole, mp 159-160°was obtained by recrystallization from methanol.

EXAMPLE 14 3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-methylpyrazole

A stirred solution of ethyl3-(3-cyclopentyloxy-4-methoxybenzylamino-pyrazole-4-carboxylate) (fromEXAMPLE 12) (5.4 g, 0.015 mol) in THF (50 mL) at 0° was treated withsodium bis (2-methoxyethoxy)aluminum hydride (0.03 mole), 9 mL of 3.4 Msolution in toluene. After 3 hours, another 9 mL portion of the sodiumbis (2-methoxy-ethoxy)aluminum hydride solution (0.03 mol) was added.The reaction was stirred overnight at room temperature and methanol (20mL) was added. The reaction. mixture was diluted with water andextracted with ethyl acetate. The ethyl acetate extracts were washedwith water and evaporated under reduced pressure. The residuecrystallized upon trituration with hexanes. Pure title compound (1.7 g,38%), mp 76.-770, was obtained after two crystallizations from methylt-butylether.

EXAMPLE 153-(3-cyclopentyloxy-4-methoxybenzylamino)-4-methoxymethylpyrazole

A solution of3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-hydroxymethylpyrazole (1.0g, 3.15 mmol) and p-toluenesulfonic acid monohydrate (0.2 g, 1.0 mmol)in methanol (100 mL) was kept at room temperature for 10 min. Thereaction mixture was neutralized with 1 N aqueous sodium hydroxide andevaporated under reduced pressure at room temperature. The residue-waspartitioned between water (50 mL) and ethyl acetate (50 mL). The layerswere separated and the aqueous layer was extracted with ethyl acetate(10 mL). The combined ethyl acetate layers were washed with water andevaporated under reduced pressure. The residue was taken up in a smallvolume of methylene chloride and applied to a 4 mm silica gel disk on aChromatotron. Elution with 525 mL of methylene chloride and 275 mL of 1%methanol/methylene chloride removed some impure material. Continuedelution with 75 mL of 1% methanol/methylene chloride afforded 380 mg(36%) of pure3-(3-cyclopentyloxy-4-methoxybenzylamino)-4-methyoxymethylpurazole, mp.112-115°.

EXAMPLE 16 ethyl 3-(3-cyclopentyloxy-4-methoxyphenyl-1-ethen-2-yl)pyrazole-4-carboxylate

A four-step process as set forth below was used to prepare the titlecompound.

A: Ethyl 5-(3-cyclopentyloxy-4-methoxyphenyl)-3-oxo-5-hydroxypentanoate

Sodium hydride (6.0 g of 60% sodium hydride in mineral oil, 0.15 mol)was placed in a 3-neck flask equipped with a mechanical stirrer undernitrogen and washed twice with hexanes. THF (200 mL) was added and themixture was cooled in an ice bath. Ethyl acetoacetate (17.6 g, 0.135mol) was added dropwise and the mixture was stirred for 10 minutes afterthe addition. Butyllithium (0.35 mol, 84 mL of 1.6 M solution inhexanes) was added dropwise to give an orange solution. A solution of3-cyclopentyloxy-4-methoxybenzaldehyde (29.6 g, 0.135 mol) in THF (100mL) was added dropwise. After 10 minutes, the cold solution was treatedwith concentrated hydrochloric acid (26 mL, 0.29 mol), water (65 mL) andether (500 m;). The ether layer was separated and washed with saturatedbrine (3×300 mL). The ether was evaporated to give 46.5 g of crude titlecompound. The crude product was dissolved in a small amount of 30%methylene chloride/hexanes and purified by chromatography. over 400 g offlash chromatography silica gel. Elution with methylene chloride/hexanemixtures removed 9 g of starting aldehyde. Elution with 1%methanol/methylene chloride afforded the ethyl.5-(3-cyclopentyloxy-4-methoxyphenyl)-3-oxo-5-hydroxypentanoate (25.0 g,53%) as an oil.

B. Ethyl 5-(3-cyclonentyloxy-4-methoxyphenyl)-3-oxo-4-pentenoate

To a solution of p-toluenesulfonic acid monohydrate (0.13 g, 0.68 mmol)warmed on the steam bath was added ethyl5-(3-cyclopentyloxy-4-methoxy)-3-oxo-5-hydroxypentanoate (13.5 g, 0.039mol). After 5 minutes, the solution was cooled and purified bychromatography over flash chromatography silica gel (130 g). Elutionwith methylene chloride/hexane mixtures gave 3.5 g (27%) of pure ethyl5-(3-cyclopentyloxy-4-methoxyphenyl)-3-oxo-4-pentenoate.

C. Ethyl5-(3-cyclopentyloxy-4-methoxyphenyl)-3-oxo-2-ethyoxymethylene-4-pentenoate

A solution of ethyl 5-(3-cyclopentyloxy-4-methoxyphenyl)3-oxo-4-pentenoate (2.2 g, 6.8 mmoo) and diethoxymethyl acetate (1.1 g,6.8 mmol) was heated at 160° for 1 hour. The reaction mixture was takenup in methylene chloride and washed with saturated sodium bicarbonatesolution. The methylene chloride was evaporated under reduced pressureand the residue was purified by flash chromatography over 15 g of flashchromatography silica gel. Elution with methylene chloride/hexanemixtures removed some starting material and by-products. Elution withpure methylene chloride afforded 1.1 g (42%) of ethyl5-(3-cyclopentyloxy-4-methoxyphenyl)-3-oxo-2-ethoxymethylene-4-pentenoate.

D. Ethyl3-(3-cyclopentyloxy-4-methoxyphenyl-ethyl-2-yl)pyrazole-4-carboxylate

A solution of ethyl5-(3-cyclopentyloxy-4-methoxyphenyl)-3-oxo-2-ethoxymethylene-4-pentenoate(1.1 g, 2.6 mmol) in ethanol (10 mL) was treated with anhydroushydrazine (0.1 g, 3.1 mmol). After standing overnight, the solvent wasremoved under reduced pressure and the residue was purified-by flashchromatography over 10 g of flash chromatography silica gel. Elutionwith methylene chloride afforded the ethyl3-(3-cyclopentyloxy-4-methoxyphenyl-ethyl-2-yl)pyrazole-4-carboxylate(0.5 g, 54%). Pure title compound mp 97-100° was obtained bycrystallization from ether/hexanes.

EXAMPLE 17 3-(3-cyclopentyloxy-4-methoxy-phenyl-1-ethen-2-yl)pyrazole-4-methanol

A solution of ethyl3-(3-cyclopentyloxy-4-methoxyphenyl-1-ethen-2-yl)pyrazole-4-carboxylate(from Example 16), (0.5 g, 1.4 mmol) in THF (50 mL) was stirredovernight with lithium aluminum hydride (0.16 g, 2.1 mmol). The reactionmixture was then treated successively with water, (0.5 mL), 15% aqueoussodium hydroxide (0.5 mL) and water (1.5 mL). The inorganic material wasfiltered and washed with methylene chloride. The ether and methylenechloride solutions were combined and washed with water and evaporated.The crude product was dissolved in methylene chloride and purified byflash chromatography over 4.5 g of flash chromatography silica gel.Elution with methylene chloride/methanol mixtures afforded3-(3-cyclopentyloxy-4-methoxyphenyl-1-ethen-2-yl)pyrazole-4-carbinol(0.30 g, 68%). Pure title compound, mp 121-123°, was obtained bycrystallization from toluene.

EXAMPLE 18 ethyl3-[2-(3-cyclopentyloxy-4-methox-phenyl)ethyl]pyrazole-4-carboxylate

A solution of ethyl3-(3-cyclopentyloxy-4-methoxyphenyl-1-ethylene-2-yl)pyrazole-4-carboxylate(Example 16), (1.1 g, 3.1 mmol) in ethyl acetate (50 mL) washydrogenated at 20 psi over 0.5 g of 10% Pd on carbon catalyst. After 24hours, the catalyst as filtered and the solution was hydrogenated againwith 0.5 g of fresh 10% Pd on carbon catalyst. After hydrogen uptakeceased,the catalyst was filtered and the crude product was purified byflash chromatography over 20 g of flash chromatography silica gel.Elution with methylene chloride and 0.25% ethanol/methylene chlorideremoved some impurities. Elution with 2% ethanol/methylene chlorideafforded ethyl3-[2-(3-cyclopentyl-oxy-4-methoxyphenyl)ethyl]pryazole-4-carboxylate(0.70 g, 63%) Pure title compound, mp 80-82° was obtained bycrystallization from ether/hexanes.

EXAMPLE 193-[2-(3-Cyclopentyloxy-4-methoxyrhenyl)ethyl]pyrazole-4-methanol

A solution of ethyl3-[2-(3-cyclopentyloxy-4-methoxyphenyl)ethyl]pyrazole-4-carboxylate(from Example 18), (0.4.0 g, 1.1 mmol) was stirred overnight withlithium aluminum hydride (0.18 g, 4.7 mmol). THF (25 mL) was added andstirring was continued for 1 hour. The reaction mixture was then treatedsuccessively with water (0.2 mL), 15% aqueous sodium hydroxide (0.2 mL)and water (0.6 mL). The inorganic material was filtered and washed andmethylene chloride. The organic solutions were combined and evaporatedunder reduced pressure. The crude product was purified by flashchromatography over 3.5 g of flash chromatography silica gel. Elutionwith methylene chloride and 1% methanol/methylene chloride removed someimpurities. Elution with 2% methanol/methylene chloride gave 0.21 g(60%) of 3-[2-(3-cyclopentyloxy-4-methoxyphenyl)ethyl]pyrazole carbinol.Pure title compound, mp 88-91°, was obtained by crystallization fromether/hexanes.

EXAMPLE 202-(3-Cyclopentyloxy-4-methoxyphenyl)-4-trifluoromethyl-imidazole

1,1-Dibromo-3,3,3-trifluoroacetone (2.95 g, 1.64 ml, 10.9 mmol) wasadded to a solution of NaOAc (1.52 g, 18 mmol) in water (8 ml). Thesolution was heated at 80° C. for 45 min. To the cooled solution at 0°C. was added 3-cyclopentyloxy-4-methoxybenzaldehyde (2 g, 9.07 mmol) andmethanol (34 ml), followed by concentrated NH₄OH (11 ml). A homogeneousreaction mixture was obtained, which was stirred overnight at roomtemperature. Volatiles were removed in vacuo and flash chromatography(SiO₂, EtOAc:hexane (3:7)) produced2-(3-cyclopentyloxy-4-methoxyphenyl)-4-trifluoromethyl imidazole (1.728g, 58%) as a white solid, mp 157-159° C.

δ_(H) (250 MHz;CDCl₃) 1.45-1.9 (8H,m,4×CH₂), 3.85 (3H,s,OMe), 4.7(1H,m,CH), 6.8 (1H,d,ArH), 7.3 (1H,d,ArH), 7.4 (2H,m,ArH), 10.3(1H,s,NH).

EXAMPLE 21 2-(3-Cycloventyloxy-4-methoxyphenyl)-imidazole-4-carbogylicacid

2-(3-cyclopentyloxy-4-methoxyphenyl)-4-trifluoromethyl imidazole (688mg, 2.11 mmol) and 1N NaOH (25 ml) were heated at 90° C. for 2 h. Afterwhich time the reaction was made acidic by addition of c.HCl, theprecipitate was collected by filtration and the filter was washed withethanol (5 ml) and ether (5×20 ml), dried in vacuo, to afford2-(3-cyclopentyloxy-4-methoxyphenyl)-imidazole-4-carboxylic acid (356mg, 56%) as a light tan solid, mp 206-208° C.

δ_(H) (250 MHz;d₄,MeOH) 1.7 (2H,m,CH₂), 1.8-2.0 (6H,m,3×CH₂), 3.9(3H,s,OMe), 4.9 (1H,m,CH), 7.1 (1H,d,ArH), 7.6 (2H,m,ArH), 8.0(1H,s,ArH).

EXAMPLE 22 2-(3-Cyclopentyloxy-4-methoxyphenyl)-imidazole-4-formamide

A: 2-(3-Cyclopentyloxy-4-methoxyphenyl)-4-cyanoimidazole

A mixture of2-(3-cyclopentyloxy-4-methoxyphenyl)-4-trifluoromethyl-imidazole (1.04g, 3.18 mmol) and 5% aqueous ammonium hydroxide (300 ml) was heated toreflux for 24 hours. The reaction mixture. was cooled and carefullyneutralised with acetic acid. The mixture was extracted with methylenechloride (3×150 ml) and the volatiles removed in vacuo to afford thecrude product. Flash chromatography (SiO₂; EtOAc:hexane (2:3)) producedthe 2-(3-cyclopentyloxy-4-methoxyphenyl)-4-cyanoimidazole (320 mg, 35%)as a pale yellow solid.

B: 2-(3-Hydroxy-4-methoxyphenyl)-4-imidazole-4-formamide

A mixture of the 2-(3-Cyclopentyloxy-4-methoryphenyl)-4-cyanoimidazole(320 mg, 1.18 mmol) and c.HCl (25 ml) was heated to 50° C. overnight.The reaction was then cooled to 0° C., neutralised with c.NH₄OH andextracted with ethyl acetate (5×80 ml), the combined organics were driedover Na₂SO₄ and the volatiles removed in vacuo. Flash chromatography(SiO₂;CH₂Cl₂ to CH₂Cl₂:EtOH (7:3) (1% NH₃)) afforded2-(3-hydroxy-4-methoxyphenyl)-imidazole-4-formamide (110 mg, 40%) as awhite solid.

C: 2-(3-Cycloventyloxy-4-methoxyphenyl)-imidazole-4-formamide

A mixture of 2-(3-hydroxy-4-methoxyphenyl)-imidazole-4-formamide (110mg, 0.47 mmol), cyclopentyl bromide (60 μl, 0.56 mmol) and K₂CO₃ (162mg, 1.2 mmol) in DMF (10 ml) were heated at 70° C. overnight. A furtherquantity of cyclopentyl bromide (60 μl, 0.56 mmol) and K₂CO₃ (162 mg,1.2 mmol) were added and the reaction mixture was cooled to roomtemperature, diluted with water (50 ml), extracted with ethyl acetate(2×100 ml). The combined organics were washed with brine (30 ml) driedover Na₂SO₄ and the volatiles removed in vacuo. Flash chromatography(SiO₂; CH₂Cl₂:EtOH (9:1) (1% NH₃)) afforded2-(3-cyclopentyloxy-4-methoxyphenyl)-imidazole-4-formamide (43 mg,) as awhite solid, mp d.>195° C.

δ_(H) (250 MHz;d₄,MeOH) 1.65 (2H,m,CH₂), 1.7-2.0 (6H,m,3×CH₂), 3.9(3H,s,OMe), 7.0 (1H,d,ArH), 7.5 (2H,m,ArH), 7.7 (1H,s,ArH).

EXAMPLE 23 2-(3-gyclopentyloxy-4-methoxphenyl)-imidazole-4-carboxylicacid-2,-6-dimethylphenyl amide

A mixture of 2-(3-cyclopentyloxy-4-methoxyphenyl)-imidazole-4-carboxylicacid (400 mg, 1.32 mmol) and SOCl₂ (10 ml) containing DMF (1 drop) washeated to reflux for 2 hours. The volatiles were removed in vacuo and tothe residue was added toluene (25 ml) and the solvent evaporated. Thisprocedure was repeated to remove dissolved gaseous by-products. Theresidue was dissolved in THF (10 ml) and added dropwise at 0° C. over 10minutes, to a solution of the salt of 2,6-dimethylaniline (510 μl, 4.14mmol) in THF (30 ml) (formed by adding a solution of the aniline in THF(15 ml) to a suspension of NaH (167 mg, 4.14 mmol) is THF (15 ml) atroom temperature). The resultant solution was stirred at roomtemperature for 48 hours, diluted with iN HCl (20 ml) and the mixtureextracted with methylene chloride (2×50 ml). The combined organics weredried over Na₂SO₄ and the volatiles removed in vacuo. Flashchromatography (SiO₂; CH₂Cl_(2:)EtOH (97:3)) afforded2-(3-cyclopentyloxy-4-methoxyphenyl)-imidazole-4-carboxylicacid-2,6-dimethylphenyl amide (80 mg, 15%) as a pale yellow solid, mp118-122° C.

δ_(H) (250 MHz;CDCl₃) 1.6 (2H,m,CH₂), 1.7-2.0 (6H,m,3×CH₂), 2.3(6H,s,2×Me), 3.9 (3H,s,OMe), 5.0 (1H,m,CH), 6.8 (1H,d,ArH), 7.1(2H,d,ArH), 7.8-8.0 (3H,m,ArH), 8.5 (1H,6s,NH), 10.3 (1H,6s,NH).

EXAMPLE 24 2-(3-Cyclopentyloxy-4-methoxyphenyl)-imidazole-4-methanol

A: 2-(3-Cyclopentyloxy-4-methoxyphenyl)-imidazole-4-methylcarboxylate

To a solution of2-(3-cyclopentyloxy-4-methoxyphenyl)-imidazole-4-carboxylic acid (0.5 g,1.65 mmol) in dry methanol (2 ml) at 0° C. was added acetyl chloride(0.8 ml, 11.2 mmol). The reaction mixture was stirred at roomtemperature, for 4 days and then the volatiles were removed in vacuo.The oily residue was dissolved in water (20 ml), basified with lN NaOHand extracted with methylene chloride (3×50 ml). The combined organicswere dried over Na₂SO₄ and the volatiles removed in vacuc. Flashchromatography (SiO₂; EtOAc:Hexane (4:1)) afforded2-(3-cyclopentyloxy-4-methoxyphenyl)-imidazole-4-methyl carboxylate (225mg, 43%), as a white solid, mp 165-168° C.

B: 2-(3-Cyclopentyloxy-4-methoxMchenyl)-imidazole-4-methanol

To a solution of2-(3-cyclopentyloxy-4-methoxyphenyl)-imidazole-4-methylcarboxylate (225mg, 0.71 mmol) in dry THF (15 ml) was added, with stirring at −40° C., asolution of diisobutylaluminium hydride (1M in THF, 6.0 ml, 60 mmol).After 1.5 hours the reaction mixture was poured onto saturated NH₄Cl (50ml) and extracted with ethyl acetate (3×100 ml). The combined organicswere washed with saturated NH₄Cl (50 ml), dried over Na₂SO₄ and thevolatiles removed in vacuo. Flash chromatography (SiO₂; CH₂Cl₂EtOH(95:5) (1% NH₃)) afforded2(3-cyclopentyloxy-4-methoxyphenyl)-imidazole-4-methanol (88 mg, 45%) asa white solid, mp 188-1900° C.

δ_(H) (250 MHz;CDCl₃) 1.5 (2H,m,CH₂), 1.6-1.9 (6H,m,3×CH₂), 3.7(3H,s,OMe), 4.5 (2H,s,CH₂O), 4.7 (1H,m,CH), 6.7 (1H,d,ArH), 6.8(1H,s,ArH), 7.3 (1H,dd,ArH), 7.4 (1H,d,ArH).

EXAMPLE 25 2-(3-cyclopentylogy-4-methoxyphenyl)-imidazole-4-carboxylicacid, N-(2-hydroxyethyl) amide

To a solution of2-(3-Cyclopentyloxy-4-methoxyphenyl)-imidazole-4-carboxylic acid (500mg, 1.65 mmol) in DMF (15 ml) at room temperature was addedcarbonyldiimidazole (295 mg, 1.8 mmol). The resultant solution wasstirred for 30 minutes and then ethanolamine (180 μl, 1.8 mmol) wasadded and the reaction mixture stirred at room temperature overnight.Water (75 ml) was added and the mixture extracted with ethyl acetate(2×75 ml). The combined organics were washed with brine (40 ml), driedover Na₂SO₄ and the volatiles removed in vacuo. Flash chromatography(SiO₂; CH₂Cl₂:EtOH (95:5) (1% NH₃)) afforded2-(3-cyclopentyloxy-4-methoxyphenyl)-imidazole-4-carboxylic acid,N-(2-hydroxyethyl) amide (247 mg, 43%) as a white solid, mp 206-208° C.

δ_(H) (250 MHz;d₆,DMSO) 1.6 (2H,m,CH₂), 1.65 (4H,m,CH₂), 1.9 (2H,mCH₂),3.3 (2H,m,NCH₂), 3.3 (1H,s,NH), 3.5 (2H,m,OCH₂), 3.8 (3H,s,OMe), 4.8(1H,m,CH), 4.8 (1H,s,OH), 7.0 (1H,d,ArH), 7.5 (1H,d,ArH), 7.7(1H,s,ArH), 7.85 (1H,t,ArH), 12.8 (1H,6s,NH).

EXAMPLE 26 4-(1-(3-Cyclopentyloxy-4-methoxMihenyl)−2-phenyl-ethyl)-5-methyl-pyrazol-3-one

A: Ethyl-3(3-Cyclopentyloxy-4-methoxyphenyl)-2-acetyl-prop-2-enoate

A mixture of 3-Cyclopentyloxy-4-methoxybenzaldehyde (7 g, 31.8 mmol),ethyl acetoacetate (4.13 g, 31.8 mmol), piperidine (275 mg, 3.18 mmol)and glacial acetic acid (100 mg) in toluene (350 ml) was heated atreflux with a Dean-Stark trap for 2 days, during which time, at regularintervals, further additions of piperidine (4×275 mg) and glacial aceticacid (4×100 mg) were made. The reaction mixture was then-diluted withwater and extracted with methylene chloride (3×150 ml), dried overNa₂SO₄ and the volatiles removed in vacuo. Flash chromatography (SiO₂;EtOAc:hexane (1:9))affordedethyl-3-(3-cyclopentyloxy-4-methoxyphenyl.)-2-acetyl-prop-2-enoate(2 g, 19%) as a yellow oil.

B: Ethyl-3-(3-Cycloventyloxy-4-methoxyphenyl)−2-acetyl-4-phenyl-butanoate

Cuprous iodide (1.15 g, 6.05 mmol) in dry THF (10 ml) was treated at 0°C. with benzylmagnesium bromide (2M in THF, 6.05 ml, 12.1 mmol) over a 3minute period. The cuprate was stirred at 0° C. for 30 minutes and thencooled to −78° C. and then TMSCl (3.28 g, 30.2 mmol) in THF (5 ml) wasadded dropwise keeping the reaction temperature below −60° C. Themixture was stirred at below −60° C. for a further 10 minutes and thenethyl-3-(3-cyclopentyloxy-4-methoxyphenyl)-2-acetyl-prop-2-enoate (1 g,3.01 mmol) in THF (5 ml) was added dropwise over 3 minutes. The mixturewas stirred at below −60° C. for 45 minutes and then saturated NH₄Cl (30ml) and water (20 ml) were added. The mixture was allowed to warm toroom temperature, the resultant precipitate was removed by filtrationand the filtrate evaporated, the residue was dissolved in methylenechloride (200 ml), washed with water (30-ml), 1n HCl (50 ml) and water(30 ml), dried over Na₂SO4 and the volatiles removed in vacuo. Flashchromatography (SiO₂; EtOAc:hexane (1:9)) affordedethyl-3-(3-cyclopentyloxy-4-methoxyphenyl)-2-acetyl-4-phenyl-butanoate(1.19 g), 92%) as a yellow oil.

C:4-(1-(3-Cyclopentyloxy-4-methoxyphenyl))-2-phenyl-ethyl)-5-methyl-pyrazol-3-one

Hydrazine monohydrate (207 mg, 4.2 mmol) was added to a solutionof3-ethyl-(3-cyclopentyloxy-4-methoxyphenyl)-2-acetyl-4-phenyl-butanoate(1.186 g, 2.8 mmol) in methanol at 0° C. The resultant mixture wasallowed to reach room temperature over 45 minutes and then heated toreflux overnight. The volatiles were removed in vacuo and flashchromatography (SiO₂; CH₂Cl₂:EtOH (95:5) (1% NH₃)) afforded4-(1-(3-cyclopentyloxy-4-methoxyphenyl)-2-phenyl-ethyl)-5-methyl-pyrazol-3-one(460 mg, 42%) as a pale yellow solid, mp 88-90° C.

δ_(H) (250 MHz;CDCl₃) 1.5 (2H,m,CH₂), 1.6-1.9 (6H,m,3×CH₂), 1.9(3H,s,Me), 3.2-3.5 (2H,m,ArCH₂), 3.8 (3H,s,OMe), 3.85 (1H,m,ArCH), 4.7(1H,m,CH), 6.7 (1H,d,ArH), 6.85 (1H,dd,ArH), 7.0 (1H,d,ArH), 7.2(5H,m,ArH).

EXAMPLE 27

Protocols for PDE IV, PDE III, and PDE V inhibition activity are setforth below:

Type III Phosphodiesterase Enzyme Isolation Protocol

The Type III PDE is isolated from human platelets using a proceduresimilar to that previously described by Weishaar, R. E.; Burrows, S. D.;Kobylarg, D. C., Quade, N. M.; Evans, D. B., Biochem. Pharmacol.,35:787, 1986. Briefly, 1-2 units of platelets are suspended in an equalvolume of buffer (20 mM Tris-HCl, pH 7.5, containing 2 mM magnesiumacetate, 1 mM dithiothreitol, and 5 mM Na₂EDTA). The proteinaseinhibitor phenylmethyl-sulfonyl fluoride (PMSF) is also included in thisbuffer at a final concentration of 200 μM. The suspension is homogenizedusing a polytron and the homogenate centrifuged at 100,000×g for 60minutes. This and all subsequent procedures are performed at 0-4° C. Thesupernatant is then filtered through four layers of gauze and applied toa DEAE-Trisacryl M column, previously equilibrated with buffer B (20 mMTris-HCl, pH 7.5, containing 1 mM magnesium acetate, 1 mM dithiothreitoland 200 μM PMSF). After application of the sample, the column is washedwith several bed volumes of buffer B, after which the different forms ofPDE are eluted from the column using two successive linear NaClgradients (0.05-0.15M, 300 ml total; 0.15-0.40M, 200 ml total). Five mlfractions are collected and assayed for cyclic AMP and cyclic GMP PDEactivity. Fractions containing PDE III activity are pooled and dialyzedovernight against 4 L of buffer B. Tbe dialyzed PDE III is thenconcentrated to 10% of the original volume, diluted to 50% with ethyleneglycol monoethyl ether and stored at −20° C. PDE III can typically beretained for up to four weeks with little or no loss of activity.

Measuring Type III PDE Activity

Enzyme activity is assessed by measuring the hydrolysis of [³H]-cyclicAMP, as described by Thompson, W. J., Teraski, W. L., Epstein, P. N.,Strada, S. J.: Adv. Cyclic Nucleotide Res. 10:69, 1979. The cyclic AMPconcentration used in this assay is 0.2 μM, which approximates to theK_(m) value. Protein concentration is adjusted to ensure that no morethan 15% of the available substrate is hydrolyzed during the incubationperiod.

All test compounds are dissolved in dimethyl sulfoxide (finalconcentration of 2.5%). This concentration of dimethyl sulfoxideinhibits enzyme activity by approximately 10%.

Type IV Phosphodiesterase Enzyme Isolation Protocol

The Type IV PDE is isolated from bovine tracheal smooth muscle using aprocedure similar to that previously described by Silver, P. J., Hamel,L. T., Perrone, M. H. Bentley, R. G. Bushover, C. R., Evans, D. B.: Eur.J. Pharmacol. 150:85,1988. (1). Briefly, smooth muscle from bovinetrachea is minced and homogenized using a polytron in 10 volumes of anextraction buffer containing 10 mM Tris-acetate (pH 7.5), 2 mM magnesiumchloride, 1 mM dithiothreitol and 2,000 units/ml of aprotinin. This andall subsequent procedures are performed at 0-4° C. The homogenate issonicated and then centrifuged at 48,000×g for 30 minutes. The resultingsupernatant is applied to a DEAE Trisacryl M column previouslyequilibrated with sodium acetate and dithiothreitol. After applicationsof the sample, the column is washed with sodium acetate/dithiothreitol,after which the different forms of PDE are eluted from the column usinga linear Tris-HCl/NaCl gradient. Fractions containing Type IV PDE arecollected, dialyzed and concentrated to 14% of the original volume. Theconcentrated fractions are diluted to 50% with-ethylene glycol andstored at −20° C.

Measuringc Type IV PDE Activity

Enzyme activity is assessed by measuring the hydrolysis of [³H]-cyclicAMP, as described by Thompson, W. J., Teraski, W. L., Epstein, P. N.,Strada, S. J.: Adv. Cyclic Nucleotide Res. 10:69, 1979. The cyclic AMPconcentration used in this assay is 0.2 μM, which approximates to theK_(m) value. Protein concentration is adjusted to ensure that no morethan 15% of the available substrate is hydrolyzed during the incubationperiod.

All test compounds are dissolved in dimethyl sulfoxide (finalconcentration of 2.5%). This concentration of dimethyl sulfoxideinhibits enzyme activity by approximately 10%.

Type V Phosphodiesterase Enzyme Isolation Protocol

Enzyme Isolation Procedure: The Type V PDE is isolated using a proceduresimilar to that previously described by Weishaar et al. (Weishaar, R.E., Kobylarz-Singer, D. C., Keiser, J., Haleen, S. J., Major, T. C.,Rapundalo, S., Peterson, J. T., Panek, R.: Hypertension 15:528, 1990).Briefly, 1-2 units of platelets are suspended in an equal volume ofbuffer A (20 mM Tris-HCl, pH 7.5, containing 2 mM magnesium acetate, 1mM dithiothreitol, and 5 mM Na₂EDTA) using a polytron. The proteinaseinhibitor phenylmethylsulfonyl fluoride (PMSF) are also included in thisbuffer at a final concentration of 200 uM. This and all subsequentprocedures are performed at 0-4° C. The homogenate is then centrifugesat 100,000×g for 60 minutes. The supernatant is then removed andfiltered through four layers of gauze and applied to a DEAE-Trisacryl Mcolumn. The column is washed with several bed volumes of buffer B (20 mMTris-HCl, pH 7.5, containing 2 mM magnesium acetate, 1 mMdiothiothreitol, and 200 uM PMSF) and eluted by two successive linearNaCl gradients (0.05-0.15 M, 300 ml total; 0.15-0.40 M, 200 ml total).Five ml fractions are collected and assayed for cyclic AMP and cyclicGMP PDE activity. Fractions that contain PDE V are pooled and dialyzedovernight against 4 L of buffer C (20 mM Tris-HCl, pH 7.5, containing 2mM magnesium acetate and proteinase inhibitors). The dialyzed PDE V isthen concentrated to 10% of the original volume, diluted to 50% withethylene glycol monoethyl ether and stored at −20° C. PDE V cantypically be retained for up to four weeks with little or no loss ofactivity.

Measuring Type V PDE Activity: Enzyme activity are assessed by measuringthe hydrolysis of [³H]-cyclic GMP, as described by Thompson et al.(Thompson, W. J., Teraski, W. L., Epstein, P. N., Strada, S. J.: Adv.Cyclic Nucleotide Res. 10:69, 1979). The cyclic GMP concentration usedin this assay is 0.2 uM, which approximates to the K_(m) value. Proteinconcentration is adjusted to ensure that no more than 15% of theavailable substrate is hydrolyzed during the incubation period.

All test compounds are dissolved in dimethyl sulfoxide (finalconcentration of 2.5%). This concentration of dimethyl sulfoxideinhibits enzyme activity by approximately 10%. The reference Type V PDE.inhibitor zaprinast is evaluated with each assay.

The compounds are tested over concentration range: 0.1, 1, 10, 100 uM(n=1), and I₅₀ determinations are made using 5 appropriateconcentrations (n=2).

EXAMPLE 28

Following the above procedures, the PDE III,. PDE IV, PDE V inhibitionfor the compound of Example 1, 3-(3-cyclopentyloxy-4-methoxybenzylamino)−4-hydroxymethylpyrazole, and rolipram (PDE III, PDE IV) are tested andcompared. The results are shown in Tables 1-3 below:

TABLE 1 PDE III ACTIVITY Molecular % Inhibition Compound Weight 1.0 μM10 μM 100 μM 300 μM EX. 1 317.4 8% 22% 53% precip- itate Rolipram — — 7% 18% 35%

TABLE 2 PDE IV ACTIVITY Molecular % Inhibition Example Mol. Wt. 0.001 μM0.003. μM 0.01 μM 0.03 μM 0.1 μM 1.0 μM 10 μM Ex. 1 317.4 10% 26% 42%58% 70% Rolipram 36% 67%

TABLE 2 PDE IV ACTIVITY Molecular % Inhibition Example Mol. Wt. 0.001 μM0.003. μM 0.01 μM 0.03 μM 0.1 μM 1.0 μM 10 μM Ex. 1 317.4 10% 26% 42%58% 70% Rolipram 36% 67%

The concentration of Example 1 which yielded 50% inhibition of PDE IV(IC₅₀) was 0.016 μM.

The concentration of rolipram which yielded 50% inhibition of PDE IV(IC₅₀) in this same assay was 4.5 μM.

EXAMPLE 29

In this Example, the compounds prepared in the above examples wereprepared as set forth above and tested for Type III and Type IV PDEActivity-in similar fashion as set forth with regard to the proceduresset forth in Example 20. The results are set forth in Table 4 below:

TABLE 4 ACTIVITIES EXAMPLE PDE IV IC₅₀ (μM) PDE III IC₅₀ (μM) 10.016 >100 2 53 >300 3 109 >300 4 4.4 >300 5 15 >100 6 20% @ 100 μM >1007 19% @ 100 μM >100 8 25 46 9 13 >100 10 7.8 >300 11 78 >300 12 3.59385.317 13 22.80 >100 14 7.30 >300 15 0.17 >300 16 1.47 7.320 178.00 >100 18 12.50 >100 19 17.508 >300 20 14.68 >100 21 7.44 >250 22 3.976 23 7.28 >200 24 17.54 >300 25 4.48 >250 26 2.80 >100

AS can seen from the foregoing, the inventive compounds provide highlevels of PDE-IV inhibition while at the same time relatively low levelsof PDE-III inhibition.

While the invention has been illustrated with respect to the productionand use of a particular compound, it is apparent that variations andmodifications of the invention can be made without departing from thespirit or scope of the invention.

What is claimed is:
 1. A compound of the formula:

wherein: X₁ and X₂ may be the same or different and each is O or S; R₁and R₂ may be the same or different and each are saturated orunsaturated straight-chain or branched alkyl groups containing from 1 to12 carbon atoms, cycloalkyl and cycloalkyl-alkyl groups containing from3 to 10 carbon atoms in the cycloalkyl moiety; or one of R₁ and R₂ arehydrogen and the other represents a hydrocarbon group as set forthabove; said R₁ and R₂ are optionally substituted with a saturated orunsaturated straight-chain lower alkyl group containing from about 1 toabout 6 carbon atoms, unsubstituted or substituted with one or morehalogen atoms, hydroxyl groups, cyano groups, nitro groups, carboxylgroups, alkoxy groups, alkoxycarbonyl, carboxamido or substituted orunsubstituted amino groups; and at least one of R₁ and R₂ is acycloalkyl moiety; R₃ is hydrogen, halogen, or a saturated orunsaturated straight-chain or branched alkyl group containing from 1 to12 carbon atoms, a cycloalkyl and cycloalkyl-alkyl groups containingfrom 3 to 7 carbon atoms in the cycloalkyl moiety; Z is a linkageselected from —CH₂—, —NH—, —CH₂O—, —CH₂CH₂—, —CH₂NH—, CH₂N(Me), NHCH₂—,—CH₂CONH—, —NHCH₂CO—, —CH₂CO—, —COCH₂—, —CH₂COCH₂—, —CH(CH₃)—, —CH═, and—HC═CH—, wherein the carbon and/or nitrogen atoms are unsubstituted orsubstituted with a lower alkyl, halogen, hydroxy or alkoxy group; R₄ isa triazole, which is unsubstituted or substituted with one or morehalogen atoms, C₁₋₄ alkyl groups, hydroxyl groups, cyano groups, nitrogroups, carboxyl groups, C₁₋₄ alkyl esters, alkoxy groups,alkoxycarbonyl, amido, carboxamido, substituted or unsubstituted aminogroups, cycloalkyl and cycloalkyl-alkyl groups containing from 3 to 10carbon atoms in the cycloalkyl moiety, aryl or aralkyl groups containingfrom about 6 to about 10 carbon atoms, or heterocyclic groups containingnitrogen, oxygen or sulfur in the ring; said alkyl, cycloalkyl,cycloalkyl-alkyl, aryl, and aryl-alkyl groups being unsubstituted orsubstituted by halogen atoms, hydroxyl groups, cyano groups, carboxylgroups, C₁₋₄ alkoxy groups, alkoxycarbonyl, carboxamnido or substitutedor unsubstituted amino groups, or one or more lower alkyl groups havingfrom 1 to 3 carbon atoms.
 2. The compound of claim 1 wherein R₁ iscycloaklyl of 3-6 carbon atoms, which cycloalkyl may be substituted byone or more alkyl groups or by one or more halogens, R₂ is hydrogen, oralkyl of 1-12 carbon atoms, and wherein R₃ is hydrogen, lower alkyl orhalogen.
 3. The compound of claim 2 wherein R₂ is lower alkyl.
 4. Thecompound of claim 3 wherein R₁ is cycloalkyl optionally substituted byone or more halogens.
 5. The compound of claim 1 wherein R₂ is methyl orethyl and wherein R₁ is cyclopentyl optionally substituted by R₅ asshown in the following structural formula:

wherein R₅ is hydrogen or a saturated or unsaturated straight-chainlower alkyl group containing from about 1 to about 6 carbon atoms,unsubstituted or substituted with one or more halogen atoms, hydroxylgroups, cyano groups, nitro groups, carboxyl groups, alkoxy groups,alkoxycarbonyl, carboxamido or substituted or unsubstituted aminogroups.
 6. The compound of claim 2 wherein Z is a linkage selected from—CH₂CH₂—, —CH₂NH—, CH₂N(Me), —NHCH₂—, —CH₂CONH—, and —NHCH₂CO—.
 7. Thecompound of claim 2, wherein X₁ and X₂ are O.
 8. The compound of claim 6wherein Z is —CH₂NH—.
 9. The compound of claim 1, selected from thegroup consisting of3-(3-Cyclopentyloxy-4-methoxybenzylamino)-1,2,4-triazole; and5-(3-Cyclopentyloxy-4-methoxybenzylamino)-1,2,3-triazole.
 10. A compoundof the formula:

wherein: X₁ and X₂ may be the same or different and each is O or S; R₁and R₂ may be the same or different and each are saturated orunsaturated straight-chain or branched alkyl groups containing from 1 to12 carbon atoms, cycloalkyl and cycloalkyl-alkyl groups containing from3 to 10 carbon atoms in the cycloalkyl moiety; or one of R₁ and R₂ arehydrogen and the other represents a hydrocarbon group as set forthabove; said R₁ and R₂ are optionally substituted with a saturated orunsaturated straight-chain lower alkyl group containing from about 1 toabout 6 carbon atoms, unsubstituted or substituted with one or morehalogen atoms, hydroxyl groups, cyano groups, nitro groups, carboxylgroups, alkoxy groups, alkoxycarbonyl, carboxamido or substituted orunsubstituted amino groups; R₃ is hydrogen, halogen, or a saturated orunsaturated straight-chain or branched alkyl group containing from 1 to12 carbon atoms, a cycloalkyl and cycloalkyl-alkyl groups containingfrom 3 to 7 carbon atoms in the cycloalkyl moiety; Z is a linkageselected from —CH₂—, —NH—, —CH₂O—, —CH₂CH₂—, CH₂N(Me), NHCH₂—,—CH₂CONH—, —NHCH₂CO—,—CH₂CO—, —COCH₂—, —CH₂COCH₂—, —CH(CH₃)—, and —CH═,wherein the carbon and/or nitrogen atoms are unsubstituted orsubstituted with a lower alkyl, halogen, hydroxy or alkoxy group; R₄ isa triazole, which is unsubstituted or substituted with one or morehalogen atoms, C₁₋₄ alkyl groups, hydroxyl groups, cyano groups, nitrogroups, carboxyl groups, C₁₋₄ alkyl esters, alkoxy groups,alkoxycarbonyl, amido, carboxamido, substituted or unsubstituted aminogroups, cycloalkyl and cycloalkyl-alkyl groups containing from 3 to 10carbon atoms in the cycloalkyl moiety, aryl or aralkyl groups containingfrom about 6 to about 10 carbon atoms, or heterocyclic groups containingnitrogen, oxygen or sulfur in the ring; said alkyl, cycloalkyl,cycloalkyl-alkyl, aryl, and aryl-alkyl groups being unsubstituted orsubstituted by halogen atoms, hydroxyl groups, cyano groups, carboxylgroups, C₁₋₄ alkoxy groups, alkoxycarbonyl, carboxamido or substitutedor unsubstituted amino groups, or one or more lower alkyl groups havingfrom 1 to 3 carbon atoms.
 11. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a compound of the structure ofthe formula:

wherein: X₁ and X₂ may be the same or different and each is O or S; R₁and R₂ may be the same or different and each are saturated orunsaturated straight-chain or branched alkyl groups containing from 1 to12 carbon atoms, cycloalkyl and cycloalkyl-alkyl groups containing from3 to 10 carbon atoms in the cycloalkyl moiety; or one of R₁ and R₂ arehydrogen and the other represents a hydrocarbon group as set forthabove; said R₁ and R₂ are optionally substituted with a saturated orunsaturated straight-chain lower alkyl group containing from about 1 toabout 6 carbon atoms unsubstituted or substituted with one or morehalogen atoms, hydroxyl groups, cyano groups, nitro groups, carboxylgroups, alkoxy groups, alkoxycarbonyl, carboxamido or substituted orunsubstituted amino groups; and at least one of R₁ and R₂ is acycloalkyl moiety; R₃ is hydrogen, halogen, or a saturated orunsaturated straight-chain or branched alkyl group containing from 1 to12 carbon atoms, a cycloalkyl and cycloalkyl-alkyl groups containingfrom 3 to 7 carbon atoms in the cycloalkyl moiety; Z is a linkageselected from —CH₂—, —NH—, —CH₂O—, —CH₂CH₂—, —CH₂NH—, CH₂N(Me), NHCH₂—,—CH₂CONH—, —NHCH₂CO—, —CH₂CO—, —COCH₂—, —CH₂COCH₂—, —CH(CH₃)—, —CH═, and—HC═CH—, wherein the carbon and/or nitrogen atoms are unsubstituted orsubstituted with a lower alkyl, halogen, hydroxy or alkoxy group; R₄ istriazole, which is unsubstituted or substituted with one or more halogenatoms, C₁₋₄ alkyl groups, hydroxyl groups, cyano groups, nitro groups,carboxyl groups, C₁₋₄ alkyl esters, alkoxy groups, alkoxycarbonyl,amido, carboxamido, substituted or unsubstituted amino groups,cycloalkyl and cycloalkyl-alkyl groups containing from 3 to 10 carbonatoms in the cycloalkyl moiety, aryl or aralkyl groups containing fromabout 6 to about 10 carbon atoms, or heterocyclic groups containingnitrogen, oxygen or sulfur in the ring; said alkyl, cycloalkyl,cycloalkyl-alkyl, aryl, and aryl-alkyl groups being unsubstituted orsubstituted by halogen atoms, hydroxyl groups, cyano groups, carboxylgroups, C₁₋₄ alkoxy groups, alkoxycarbonyl, carboxamido or substitutedor unsubstituted amino groups, or one or more lower alkyl groups havingfrom 1 to 3 carbon atoms.
 12. The pharmaceutical composition of claim 11which is suitable for oral administration.
 13. The pharmaceuticalcomposition of claim 11 which is suitable for parenteral administration.14. The pharmaceutical composition of claim 11 which is suitable foradministration by inhalation.
 15. The pharmaceutical composition ofclaim 11 which is suitable for administration by insufflation.
 16. Thepharmaceutical composition of claim 11 which is suitable foradministration by suppository.
 17. The pharmaceutical composition ofclaim 11 wherein said compound is selected from the group consisting of:3-(3-Cyclopentyloxy-4-methoxybenzylamino)-1,2,4-triazole; and5-(3-Cyclopentyloxy-4-methoxybenzylamino)-1,2,3-triazole.
 18. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of claim
 10. 19. A method of treating a mammalsuffering from a disease state selected from the group consisting ofasthma, allergies, inflammation, depression, dementia and disease statesassociated with abnormally high physiologic levels of cytokine,comprising administering an effective amount of a compound of theformula:

wherein: X₁ and X₂ may be the same or different and each is O or S; R₁and R₂ may be the same or different and each are saturated orunsaturated straight-chain or branched alkyl groups containing from 1 to12 carbon atoms, cycloalkyl and cycloalkyl-alkyl groups containing from3 to 10 carbon atoms in the cycloalkyl moiety; or one of R₁ and R₂ arehydrogen and the other represents a hydrocarbon group as set forthabove; said R₁ and R₂ are optionally substituted with a saturated orunsaturated straight-chain lower alkyl group containing from about 1 toabout 6 carbon atoms, unsubstituted or substituted with one or morehalogen atoms, hydroxyl groups, cyano groups, nitro groups, carboxylgroups, alkoxy groups, alkoxycarbonyl, carboxamido or substituted orunsubstituted amino groups; and at least one of R₁ and R₂ is acycloalkyl moiety: R₃ is hydrogen, halogen, or a saturated orunsaturated straight-chain or branched alkyl group containing from 1 to12 carbon atoms, a cycloalkyl and cycloalkyl-alkyl groups containingfrom 3 to 7 carbon atoms in the cycloalkyl moiety; Z is a linkageselected from —CH₂—, —NH—, —CH₂O—, —CH₂CH₂—, —CH₂NH—, CH₂N(Me), NHCH₂—,—CH₂CONH—, —NHCH₂CO—, —CH₂CO—, —COCH₂—, —CH₂COCH₂—, —CH(CH₃)—, —CH═, and—HC═CH—, wherein the carbon and/or nitrogen atoms are unsubstituted orsubstituted with a lower alkyl, halogen, hydroxy or alkoxy group; R₄ istriazole, which is unsubstituted or substituted with one or more halogenatoms, C₁₋₄ alkyl groups, hydroxyl groups, cyano groups, nitro groups,carboxyl groups, C₁₋₄ alkyl esters, alkoxy groups, alkoxycarbonyl,amido, carboxamido, substituted or unsubstituted amino groups,cycloalkyl and cycloalkyl-alkyl groups containing from 3 to 10 carbonatoms in the cycloalkyl moiety, aryl or aralkyl groups containing fromabout 6 to about 10 carbon atoms, or heterocyclic groups containingnitrogen, oxygen or sulfur in the ring; said alkyl, cycloalkyl,cycloalkyl-alkyl, aryl, and aryl-alkyl groups being unsubstituted orsubstituted by halogen atoms, hydroxyl groups, cyano groups, carboxylgroups, C₁₋₄ alkoxy groups, alkoxycarbonyl, carboxamido or substitutedor unsubstituted amino groups, or one or more lower alkyl groups havingfrom 1 to 3 carbon atoms.
 20. The method of claim 19 wherein saidcompound is selected from the group consisting of:3-(3-Cyclopentyloxy-4-methoxybenzylamino)-1,2,4-triazole; and5-(3-Cyclopentyloxy-4-methoxybenzylamino)-1,2,3-triazole.
 21. A methodof effecting selective PDE IV inhibition to a patient requiring thesame, which comprises administering an effective amount of the compoundof claim
 1. 22. The method of claim 21 wherein said compound is selectedfrom the group consisting of:3-(3-Cyclopentyloxy-4-methoxybenzylamino)-1,2,4-triazole; and5-(3-Cyclopentyloxy-4-methoxybenzylamino)-1,2,3-triazole.
 23. A methodof treating a mammal suffering from a disease state selected from thegroup consisting of asthma, allergies, inflammation, depression,dementia and disease states associated with abnormally high physiologiclevels of cytokine, comprising administering an effective amount of acompound of claim 10.